This invention relates to a novel method for decreasing the foam formation during the cultivation of Trichoderma production host, to a method for producing a product by cultivating a microorganism and to a novel production host strain with decreased foam formation during cultivation. Furthermore this invention relates to a process for producing an enhanced amount of a product of interest according to the method for decreasing the foam formation during cultivation of a Trichoderma production host.
When microorganisms are cultured in liquid growth medium in laboratory vessels or in small or large scale bioreactors, a common feature is foaming. In particular, this is a problem in aerated bioreactor or, as they are usually called, fermentor cultivations. Biochemically, the term “fermentation” refers to the process of ethanol production in yeasts by anaerobic metabolism. Modern aseptic submerged “fermentation” of individual selected microbes is used for production of cell mass, proteins such as enzymes and antibodies, and other metabolites such as antibiotics, amino acids and organic acids. The main microorganisms used in industry in fermentation are fungi, especially filamentous fungi and yeast, especially filamentous fungi and yeast, and bacteria, such as Bacillus spp., Escherichia coli and Streptomyces spp.
Typically only 70-80% of the fermentation vessel volume is filled with liquid and a gas space occupies the top portion of the tank. The combined action of aeration and agitation of the liquid promotes the formation of a foam on the liquid surface and normally all aerated fermentation broths foam, which is why a large gas space is required. Foam impedes gas mass transfer from the broth to the head space, forcing foam out of the vessel and contaminating the system when collapsed foam re-enters the fermentor. This means that some foam control method should always be included in fermentations. The two methods most commonly used for commercial fermentations are mechanical foam breakers and/or addition of antifoam agents.
A mechanical foam breaker is a high-speed impeller designed very much like a centrifugal pulp impeller. Foam is drawn into the impeller, where it is collapsed by strong mechanical forces. This is not suitable for delicate organisms. The impeller must be mounted on its own shaft and driven independently of the main agitator. This requires a separate agitator seal, which is a potential source of contamination and other problems. Mechanical foam breakers also represent a conciderable capital outlay.
Chemical antifoam agents collapse foams by altering their surface tension characteristics. Sterile antifoam agent is usually pumped into the fermentor automatically from an addition vessel. Addition of antifoam agent results in a possible contamination risk because the polymeric water-free liquid agents are difficult to sterilize. The choice of antifoam agent cannot be made only on the basis of its compatibility with the fermentation, but also with product recovery, concentration and purification in downstream processing. For example particularly silicon-based antifoam agents may decrease dramatically the permeate flux through certain types of membrane filters even at very low concentrations. Hydrophobic antifoam agents bind to the hydrophobic ultrafiltration membranes, lowering the permeate flux, and may change dramatically the apparent molecular weight cut-off of the membranes.
The first antifoam agents to be used in fermentation were silicone-based fluids. More recently, oil-based antifoam agents with a chemical structure consisting of polymers of e.g. ethylene and propylene oxides with esters of long-chain fatty acids have commonly been used. All antifoam agents have a “cloud point”, above which they are essentially insoluble in water. The functional properties of the antifoam agents operate at temperatures above the cloud point. Therefore the cloud point of the antifoam agent should be below the temperature of the fermentation. However, for example membrane filtration should be operated at temperatures below the cloud point, so that the antifoam agent will be water-soluble and therefore have minimal effect on the filtration procedure. Antifoam agents are available with very variable cloud points.
Most antifoam agents, because of their hydrophobic nature, are difficult to sterilise. They may also represent a considerable cost outlay. A good antifoam control system should also include an option to reduce automatically the air flow and the agitation speed when foaming overwhelms the system and the fermenter is in danger of being emptied. This is necessary to avoid damage to the drive system if the fermenter is emptied because of overfoaming.
Because of their function of disrupting air-filled bubbles both above and within the fermentation broth, antifoam agents considerably reduce the oxygen transfer rate. By contrast, detergents usually enhance oxygen transfer rates. When antifoam agents and detergents are present simultaneously, they exert compensating effects. Oxygen transfer rates depressed owing to the addition of antifoam agent result in decreased dissolved oxygen levels at a constant rate of agitation and aeration. Antifoam agents decrease the surface tension, resulting in reduced volumetric mass transfer coefficient (kLa) values. This leads to a need for increased aeration and agitation, which in turn enhances foam formation and necessitates further addition of antifoam agent which further enhances the problems created by antifoam agent. The positive feedback effects of foaming and agitation/aeration represent a major problem in many commercial fermentation processes. A further practical disadvantage is reduced sensitivity and accelerated ageing of probes due to clogging by the polymer components of the antifoam agents.
Production media used in industrial fermentations often contain insoluble polymers. The presence of these insoluble medium components considerably aggravates the problem of foaming during fermentation. The foam produced during cultivation combines with unutilised solid particles from the medium to form a composite foam with strong physical properties and undesirably high persistence. This foam-solids conglomerate can remain attached to the sides of the vessel and to other steel structures in the fermenter headspace independently of support from below, and contact between the broth and foam layers is disestablished. Addition of antifoam agent to the fermentation is therefore not successful in degrading the secondary foam structure. Thus after establishment of contact between the upper foam layer and the tip of the antifoam probe, the subsequent automatic addition of antifoam agent does not disrupt the foam layer. In this case, addition of antifoam agent may, and frequently does, continue indefinitely until the addition vessel becomes empty. Despite continuing addition of antifoam agent, the conglomerate foam layer continues to rise in the fermenter headspace, eventually entering the exhaust line and blocking the outlet filter. This in turn prevents the passage of air through the broth and the dissolved oxygen decreases to zero, with disastrous effects on the production process. This whole cycle may occur in a short period of only 1-2 hours.
Oxygen transfer rate is affected by the microbial species, its morphology and concentration. Species with more complex morphology (pellets versus filaments, pellets do not have significantly higher oxygen demand and uptake but clearly higher maximum oxygen transfer rate) lead to lower oxygen transfer rates. Increased viscosity due to the high mycelial concentrations (pseudoplastic moulds) concentration leads to reduced oxygen transfer rates.
Mycelial morphology also affects the process productivity and kinetics. In some cases the small pellets are optimal for the production of desired product whereas in other cases filamentous growth has been found to be optimal. The morphology also affects the downstream prossessing and filtration properties of the culture liquid as larger particles are easier to separate e.g. in vacuum drum filtration. Growth in the form of pellets usually leads to an interstitial culture fluid with greater clarity and lower viscosity than in the case of diffuse mycelial growth.
Because of the several problems caused by foam formation during fermentation there is a high demand for new ways to hinder or cut down the foam formation.
There are some prior art publications which relate to the production of surfactins in Bacillus strain bacteria. EP 576 050 describes the isolation and characterization of the sfp gene in Bacillus subtilis, which codes for a protein for producing the lipopeptide surfactin. Surfactin is a specific protein, where the lipide part is covalently bound to the protein part. Foaming properties of surfactin from B. subtilis are discussed in Razafindralambo et al. (1996) and surfactin negative phenotype of B. subtilis in D' souza et al. (1993). WO 98/22598 suggested a modification to a Bacillus cell to produce a lower level of surfactin. The modification was reported to result in reduced foaming. However, these publications were restricted to the surfactins of Bacillus. 
WO 96/41882 suggested the production of hydrofobins from edible fungi for food industry. The publication describes the overexpression of hydrophobins and does not discuss the foaming problem.